We and several of our neighbors have had problems with many GST-fusion
proteins being very difficult to elute from the GST-sepharose. Several
other of our friends say that it is no problem at all.
In general, we get 10% elution (typically) in 50 to 100 mM GSH (made
fresh, of course) pH adjusted to 8, and made from two different batches of
GSH. This particularly affects large proteins of 50 to 80 kD.
We are beginning to think that the difference is in the source of our
GST-beads. Our lab and one neighbor use some from Sigma that are about 3-5
years old (we have about $800 worth of these beads, so I'd like to be able
to use them). Our Cat # and lot number are G4510 and 119F7821. A neighbor
with similar bad results have used this cat # but a different lot number,
still pretty old. Good results have been reported with the "same" Sigma
product from about 6 months ago, and with the Pharmacia product about 6
months old too.
We are beginning to think that these beads are somehow making strong
attachments either through a bad initial manufacture, or by some aging
process. Our beads have been stored dessicated at -20. SDS/DTT boiling
elutes the proteins well, though.
I'm soliciting the experiences of you out there who either have, or do not
have this problem, to answer these short questions and return them to me.
Please don't post your individual responses to the board, unless you
really know what is going on. I'll post a summary in a few weeks.
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1) Have you had problems with poor elution from GST-beads??
2) Did you come up with a means for good elution?
3) What Source were the beads that worked well?
Manufacturer:
Cat#
lot #
approx age:
4) What Source were the beads that did not elute well?
Manufacturer:
Cat#
lot #
approx age:
5) What do you think is going on? What is your evidence?
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Thanks all for your help.
Dennis Templeton
Institute of Pathology
CWRU School of Medicine
djt2 at po.cwru.edu